No progress was made due to poor time management. Hours spent in lab were in efforts to bolster Salmonella Research Report. The following week will consist of a goal to reach 30 hours of lab time, and to finish Week 6 and Week 7 progress.
Week 7
Second attempt of gel electrophoresis for PCR Primer Overlap with D1-E14 as primers.
Agarose Gel Information:
The primers used to to synthesize the gene of Carbonic Anyhdrase - Salmonella (PDB #3QY1) were E1-D14 for a total of 14 primers.
Similar KOD PCR reagents in each PCR well included 10x rxn buffer, 25mM MgSO4, 2 mM dNTPs, dH20, and most importantly, KOD hotstart Polymerase (which must be kept cold). As seen, within the 7th well a smear appears in addition to a slight band in the 8th well. Although not as accurate of a gel as desired, it is cause to proceed on to PCR squared.
Contains a total of 663 characters (nucleotides) which is reflected along the Secondary PCR band in relation to the 100 bp ladder.
Week 6
Worked on ProtocolPCR_PrimerOverlap: consisted of PCR 1' and Second PCR; however, after removal of Gel Electrophoresis from the tray, my gel broke. When I get back into the lab, I will re-run the samples because I saved enough in case things went awry during my work. I will upload PCR picture after I re-run the cloning.
Also, my second run of Virtual Refresher is currently running.
Week 5
A continuation of PyMOL Refresher. In addition, I ran first round of Virtual Screening
Week 4
Test of PCR cloning of Insert Vector with LIC primers to get PCR product suitable for insertion into pNIC-Bsa4. PCR failed most probably during the Dilution stage.
PyMOL Refresher Results:
Trypanosoma cruzi Dihydrofolate Reductase-Thymidylate synthase (PDB identifier: 2H2Q). Chain A and Chain B are shown in red and blue, respectively. The NAP substrates for each chain are in the enzyme’s active site connected by hydrogen bonds as seen by the black dotted lines.
Bifunctional TcDHFR-TS in complex with MTX (PDB identifier: 3CL9). Chain A, colored lightorange, is seen with NAP and MTX in its active site. NAP is blue, and MTX is red. In addition, polar contacts are seen as the dotted black lines connecting the previously mentioned substrates within the enzyme’s active site (green sticks).
Magnified active sites for Human and T. cruzi DHFR.
Alignment of Human (green) and T. cruzi (blue) DHFR. Active site for Human DHFR is shown in orange, and the active site for T. cruzi is shown in hot pink. In addition, MTX1 for Human DHFR is shown in black, and MTX2 for T. cruzi DHFR is shown in grey. The RMS value for alignment is 1.126.
Pairwise comparison for human DHFR and T. cruzi DHFR active sites. As seen, only a slight variance exists.
dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate (PDB identifier: 3HBB). Chain A, red, chain B, green, chain C, blue, and chain D, yellow are shown.
Active site (cyan) of Chain A with inhibitor TMQ (black). Polar contacts are also shown as dotted black lines in the active site.
Week 3
In Progress: Submit DNA Sequencing (needs weekend to run)
PCR Run #3: This sample yielded to best results out of the 3 runs. The primer used was the Forward/Reverse M13 instead of the SP6 and T7, which may have been defective
Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the M13 Forward and Reverse primers. This adjustment was made due to the fact that the previous 2 PCR runs,
using SP6 and T7, were ineffective.
Lane 1: SKIPPED
Lane 2: 100 bp ladder
Lane 3: Sample A
Lane 4: Sample B
Lane 5: Sample C
Lane 6: Sample D
Week 2
1 kb DNA Ladder Reference
Fragments of pgbr22 plasmid cut by certain Restriction Enzymes (EcoRI, PvuII, or a combination of the both). The lanes are referenced with a 1kb DNA Ladder in Lane 2
Agarose Gel Information:
A 1 kb DNA ladder was used instead of a 100 bp ladder. In addition, each sample contains the following with only the Restriction Enzyme and corresponding Enzyme Buffer differing:
1.5 ug plasmid
2.5 ul 10X Enzyme Buffer
X ul DDW to 24ul
1 ul ~10 Units of Restriction Enzyme (or ~5 Units of each)
25 ul Total Volume
Lane 1: SKIPPED
Lane 2: 1 kb DNA ladder
Lane 3: Diluted, uncut plasmid
Lane 4: Sample A (RE-EcoRI)
Lane 5: Sample B (RE-PvuII)
Lane 6: Sample C (RE-EcoRI & PvuII)
Agarose gel results from PCR Run #2. The color is not inverted as seen in the gel of "Week 1;" however, the layered column still represents the same 100 bp DNA ladder
Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the SP6 and T7 Promoters, respectively. The second run of PCR yielded the same results, which could
suggest contamination of the SP6 and T7 Promoters (if PCR technique was flawless although that is highly unlikely).
Lane 1: SKIPPED
Lane 2: 100 bp ladder
Lane 3: Sample A
Lane 4: Sample B
Lane 5: Sample C
Lane 6: Sample D
Week 1
100 bp DNA Ladder Reference
As seen above, the first PCR run did not yield substantial results. The two dark portions are equal amounts (5ul) of the same 100 bp DNA ladder visualized by ethidium bromide staining on a TAE agarose gel
Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the SP6 and T7 Promoters, respectively.
Lane 1: SKIPPED
Lane 2: 100 bp ladder
Lane 3: Sample A
Lane 4: Sample B
Lane 5: Sample C
Lane 6: Sample D
Lane 7: SKIPPED
Lane 8: 100 bp ladder
Comments:
Johnny - add in your other gels and be sure to label everything. - Dr. B
Week 8
No progress was made due to poor time management. Hours spent in lab were in efforts to bolster Salmonella Research Report. The following week will consist of a goal to reach 30 hours of lab time, and to finish Week 6 and Week 7 progress.
Week 7
Agarose Gel Information:
The primers used to to synthesize the gene of Carbonic Anyhdrase - Salmonella (PDB #3QY1) were E1-D14 for a total of 14 primers.
Similar KOD PCR reagents in each PCR well included 10x rxn buffer, 25mM MgSO4, 2 mM dNTPs, dH20, and most importantly, KOD hotstart Polymerase (which must be kept cold). As seen, within the 7th well a smear appears in addition to a slight band in the 8th well. Although not as accurate of a gel as desired, it is cause to proceed on to PCR squared.
>gi|16763390:c202072-201410 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 chromosome, complete genome:
ATGAAAGACATAGATACACTCATCAGCAACAATGCACTATGGTCAAAAATGCTGGTGGAAGAGGACCCCGGATTTTTTGAGAAACTGGCGCAAG
CGCAAAAACCGCGCTTTCTATGGATTGGATGTTCCGACAGCCGCGTTCCCGCAGAGCGTTTAACCGGGCTTGAACCGGGCGAATTGTTTGTTCA
CCGTAATGTGGCTAACCTGGTTATTCACACCGATCTGAACTGTCTCTCCGTGGTTCAGTATGCGGTAGATGTTCTGGAAGTTGAGCATATTATCATT
TGCGGCCACTCCGGTTGCGGCGGTATCAAGGCTGCGGTAGAAAACCCTGAGCTGGGCTTGATTAATAACTGGCTGCTGCATATCCGCGACATCTG
GCTTAAACATAGCTCGCTGTTGGGAAAAATGCCCGAAGAGCAACGTCTGGACGCGCTCTATGAATTGAACGTCATGGAACAGGTCTATAACCTGG
GGCATTCCACCATTATGCAGTCAGCGTGGAAACGCGGTCAGAATGTGACCATTCACGGCTGGGCGTACAGTATCAATGATGGCCTGCTGCGCGAT
CTTGACGTCACCGCCACCAACAGAGAAACGCTGGAGAACGGCTATCATAAGGGTATTTCCGCCCTAAGTCTGAAATACATTCCCCACCAATAA
Contains a total of 663 characters (nucleotides) which is reflected along the Secondary PCR band in relation to the 100 bp ladder.
Week 6
Worked on ProtocolPCR_PrimerOverlap: consisted of PCR 1' and Second PCR; however, after removal of Gel Electrophoresis from the tray, my gel broke. When I get back into the lab, I will re-run the samples because I saved enough in case things went awry during my work. I will upload PCR picture after I re-run the cloning.Also, my second run of Virtual Refresher is currently running.
Week 5
A continuation of PyMOL Refresher. In addition, I ran first round of Virtual ScreeningWeek 4
PyMOL Refresher Results:
Week 3
In Progress: Submit DNA Sequencing (needs weekend to run)Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the M13 Forward and Reverse primers. This adjustment was made due to the fact that the previous 2 PCR runs,
using SP6 and T7, were ineffective.
Week 2
Agarose Gel Information:
A 1 kb DNA ladder was used instead of a 100 bp ladder. In addition, each sample contains the following with only the Restriction Enzyme and corresponding Enzyme Buffer differing:
Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the SP6 and T7 Promoters, respectively. The second run of PCR yielded the same results, which could
suggest contamination of the SP6 and T7 Promoters (if PCR technique was flawless although that is highly unlikely).
Week 1
Agarose Gel Information:
The Forward and Reverse Primers used to amplify the Purple Protein coding sequence in the pgbr22 plasmid
were the SP6 and T7 Promoters, respectively.
Comments:
Johnny - add in your other gels and be sure to label everything. - Dr. B